Weber M, Möller K, Welzeck M, Schorr J
QIAGEN GmbH, Hilden, Germany.
Biotechniques. 1995 Dec;19(6):930-40.
The suitability of different purification methods for preparation of plasmid DNA for transfection into eukaryotic cells was systematically investigated. The reporter plasmid, pRSVcat, was prepared using several methods, and residual impurities in the preparations were quantitated. Transfection with these preparations was performed with several cell lines (HeLa, Huh7, COS7 and LMH) and two transfection methods: liposome-mediated and calcium phosphate transfection. Transfection efficiencies were determined by measuring chloramphenicol acetyltransferase expression. Higher transfection efficiencies were obtained with plasmid preparations of higher purity (those prepared by anion-exchange chromatography or two rounds of CsCl-gradient centrifugation) than with preparations of lower purity (those prepared using a silica-based DNA adsorption method or a single round of CsCl centrifugation). The results also demonstrated specifically that increasing concentrations of lipopolysaccharides in plasmid preparations directly correlate with decreasing transfection efficiencies.
系统研究了不同纯化方法制备用于转染真核细胞的质粒DNA的适用性。使用多种方法制备报告质粒pRSVcat，并对制备物中的残留杂质进行定量。用几种细胞系（HeLa、Huh7、COS7和LMH）和两种转染方法（脂质体介导转染和磷酸钙转染）对这些制备物进行转染。通过测量氯霉素乙酰转移酶的表达来确定转染效率。与较低纯度的制备物（使用基于硅胶的DNA吸附方法或一轮CsCl离心制备的那些）相比，较高纯度的质粒制备物（通过阴离子交换色谱法或两轮CsCl梯度离心制备的那些）获得了更高的转染效率。结果还特别表明，质粒制备物中脂多糖浓度的增加与转染效率的降低直接相关。
